1. Field of the Invention
The invention in the fields of molecular biology and medicine relates to a method for detecting mutations involving as little as one base change or a single base addition to, or deletion from, the wild-type DNA sequence, as well as methods for removing mismatch-containing DNA from batches of amplified DNA.
2. Description of the Background Art
Progress in human molecular and medical genetics depends on the efficient and accurate detection of mutations and sequence polymorphisms, the vast majority of which results from single base substitutions and small additions or deletions. Assays capable of detecting the presence of a particular mutation or mutant nucleic acid sequence in a sample are therefore of substantial importance in the prediction and diagnosis of disease, forensic medicine, epidemiology and public health. Such assays can be used, for example, to detect the presence of a mutant gene in an individual, allowing determination of the probability that the individual will suffer from a genetic disease. The ability to detect a mutation has taken on increasing importance in early detection of cancer or discovery of susceptibility to cancer with the discovery that discrete mutations in cellular oncogenes can result in activation of that oncogene leading to the transformation of that cell into a cancer cell (Nishimura, S. et al., Biochem. J. 243:313-327 (1987); Bos, J. L., Cancer Res. 49:4682-4689 (1989)).
The desire to increase the utility and applicability of such assays is often frustrated by assay sensitivity as well as complexity and cost. Hence, it would be highly desirable to develop more sensitive as well as simple and relatively inexpensive assays for detection of alterations in DNA.
Nucleic acid detection assays can be based on any of a number of characteristics of a nucleic acid molecule, such as its size, sequence, susceptibility to digestion by restriction endonucleases, etc. The sensitivity of such assays may be increased by altering the manner in which detection is reported or signaled to the observer. Thus, for example, assay sensitivity can be increased through the use of detectably labeled reagents, wherein the labels may be enzymes (Kourilsky et al., U.S. Pat. No. 4,581,333), radioisotopes (Falkow et al., U.S. Pat. No. 4,358,535; Berninger, U.S. Pat. No. 4,446,237), fluorescent labels (Albarella et al., EP 144914), chemical labels (Sheldon III et al., U.S. Pat. No. 4,582,789; Albarella et al., U.S. Pat. No. 4,563,417), modified bases (Miyoshi et al., EP 119448), and the like.
Most methods devised to attempt to detect genetic alterations consisting of one or a few bases involve hybridization between a standard nucleic acid (DNA or RNA) and a test DNA such that the mutation is revealed as a mispaired or unpaired base in a heteroduplex molecule. Detection of these mispaired or unpaired bases has been accomplished by a variety of methods. Mismatches have been detected by means of enzymes (RNaseA, MutY) which cut one or both strands of the duplex at the site of a mismatch (Myers, R. M. et al., Cold Spring Harbor Symp. Quant. Biol. 51:275-284 (1986); Gibbs, R. et al., Science 236:303-305 (1987); Lu, A. S. et al., 1992, Genomics 14:249-255 (1992)). Duplexes without mismatches are not cut. By using radioactively labeled nucleic acid fragments to anneal to a test DNA, it is possible to use these enzymes to generate specific size fragments when a mutation is present in the test DNA. The fragments are distinguished from uncut fragments by means of polyacrylamide gel electrophoresis. The major problems with these methods are that they require the use of RNA (RNase method) or have the ability to detect only a limited number of mismatches (MutY method).
Mismatch-containing DNA duplexes have also been distinguished from perfectly matched duplexes by means of denaturing gel electrophoresis. In this system, duplexes are run on a polyacrylamide gel in a denaturing gradient under conditions where mismatch-containing DNA denatures more readily than the identical duplex lacking a mismatch, such that the two kinds of duplexes migrate differently. This method, while sensitive and accurate, is extremely laborious and requires a high level of technical sophistication.
Two other methods of mutation detection depend on the failure to extend or join fragments of DNA when mismatches are present. Both require the use of standard DNA oligonucleotides that end precisely at the site of the mutation in question such that, when annealed to test DNA, it is the last base of the oligonucleotide which is mismatched. Mismatch detection depends either on (a) the inability of DNA polymerase to extend an oligonucleotide with a mismatched terminal base or (b) the inability of DNA ligase to join two oligonucleotides when there is a mismatch at the joint between them. Fragment length is determined by gel electrophoresis. Presence of longer fragments than the input oligonucleotides indicates that a mismatch, i.e., mutation, was not present in the test DNA. These methods are also somewhat laborious, require that the exact location of the mutation be known and are difficult to interpret when the sample DNA is heterozygous for the mutation in question. Therefore, they are not practical for use in screening for polymorphisms.
A chemical method for cleavage of mismatched DNA (Cotton, R. G. et al., Proc. Natl. Acad. Sci. USA 85:4397-4401 (1988); Cotton, R. G., Nuc. Acids Res 17:4223-4233 (1989)) is based on chemical cleavage at a mismatch site in a DNA-DNA heteroduplex, using a number of agents, in particular osmium tetroxide and hydroxylamine. In this method DNA probes are prepared by restriction enzyme cleavage of DNA of interest. Plasmid DNA containing the sequence of interest is hybridized to labeled probe DNA (either end-labeled or internally labeled with .sup.32 P). Hydroxylamine chemically modifies mismatched cytosines; osmium tetroxide modifies mismatched thymines. Piperidine is then used to cleave the DNA at the modified sites, followed by polyacrylamide gel electrophoresis (PAGE) and autoradiography to identify the cleavage products. This method is said to have the advantage of detecting all possible single base pair mismatches because, the method also results in cleavage at a matched base pair in the vicinity of a mismatch.
Publications from Caskey's laboratory (Caskey, C. T. et al., European Patent Publication 333,465 (Sep. 20, 1989); Grompe, M. et al., Proc. Natl. Acad. Sci. USA 86:5888-5892 (1989)) disclose a method for localizing a mutation which utilizes PCR-amplified cDNA as a source of template for the mismatch cleavage reaction. This technique was successfully applied in studying ornithine transcarbamoylase (OTCase) deficiency patients to map mutations.
Kung et al., U.S. Pat. No. 4,963,658, discloses detection of single stranded DNA (ssDNA) by binding with a high-affinity ssDNA-binding protein, such as a topoisomerase or a DNA unwinding protein which itself can be bound to a label, such as .beta.-D-galactosidase.